Our long range goal is to understand the mechanism and regulation of transcription in eukaryotes. The objective for the next project period is to determine the structure of the initiation complex formed by a set of five general transcription factors and RNA polymerase II at a promoter. The work will entail molecular genetic, biochemical, electron and X-ray crystallographic studies, as well as technical development related to the electron crystallographic approach. Specific aims of the research are as follows: 1. To map the surface topography of RNA polymerase II in 2-D crystals at 16 angstrom resolution. The locations of eight of eleven subunits of the enzyme, including the C-terminal repeat domain of the largest subunit, important in regulation, will be determined. Sites of interaction with general transcription factors will be identified. DNA and RNA-binding regions will be delineated. 2. To determine the structure of RNA polymerase II at high resolution. A combination of X-ray crystallographic analysis and electron microscopy of 2-D, crystals will be employed. The structures of enzyme-DNA complexes and transcription elongation complexes, containing enzyme, DNA, and RNA will also be investigated. 3. To determine the structures of general transcription factors at high resolution. The structure of TFIID will be derived from crystals that diffract to 2.5 angstrom resolution, and the four additional general factors will be subjected to crystallographic analysis as well. 4. To determine the arrangement of transcription factors, RNA polymerase II, and promoter DNA in the initiation complex by electron microscopy and image processing of 2-D crystals. The results will be combined with those from X-ray crystallographic analysis of the individual components to arrive at a high resolution structure of the entire complex. 5. To continue development of the lipid layer crystallization approach for growing 2-D protein crystals. The range of the approach and resolution of structural information derived will be extended by engineering novel adaptors for binding proteins to lipid layers, by improving the preservation of 2-D crystals, and by conducting fundamental studies of the 2-D crystallization process.